An antimicrobial peptide capable of killing drug-resistant bacteria and its application

Abstract

The present invention relates to an antimicrobial peptide with the function of killing drug-resistant bacteria and its application, belonging to the field of biomedicine technology. The present invention is based on the antimicrobial peptide SAAP-148, and designs a new antimicrobial peptide AMP-36 by adding a partial sequence of the antimicrobial peptide Buforin2b with bacterial nucleus damage. The antimicrobial peptide has a stronger ability to kill drug-resistant bacteria and lower toxicity to normal cells than SAAP-148, laying a foundation for the development of efficient and low-toxic treatment methods for drug-resistant bacteria and non-drug-resistant bacteria infection diseases and the development of new anti-infection biological drugs.

Description

Technical Field

[0001] The invention relates to an antimicrobial peptide capable of killing drug-resistant bacteria and application thereof, belonging to the technical field of biomedicine. Background Art

[0002] Severe pneumonia caused by drug-resistant bacteria refers to a severe lung infection caused by pathogens that are resistant to multiple antibiotics. Patients often have severe symptoms such as high fever, cough, dyspnea, and hypoxia. If it cannot be effectively controlled, it will develop into toxicosis, with rapid progression and high mortality. Skin wound infection caused by drug-resistant bacteria refers to skin and soft tissue infections caused by pathogens that are resistant to multiple antibiotics. With the widespread use and abuse of antibiotics, the problem of bacterial resistance is becoming increasingly serious. Common drug-resistant bacteria include methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), carbapenem-resistant Enterobacteriaceae (CRE), and extensively drug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii. These drug-resistant bacteria are often spread in hospital environments, especially in intensive care units (ICUs). Patients usually have underlying diseases, low immune function, or a history of long-term use of broad-spectrum antibiotics, making them more susceptible to infection with drug-resistant bacteria. With the increase of antibiotic resistance, the effectiveness of traditional antibiotic treatment has been greatly reduced, the incidence of severe pneumonia and skin wound infections has increased year by year, and treatment options have become limited.

[0003] As a new type of antimicrobial treatment, antimicrobial peptides (AMPs) have gradually attracted widespread attention. Antimicrobial peptides are a class of short-chain polypeptides that exist naturally in a variety of organisms and have broad-spectrum antibacterial, antiviral, antifungal and antiparasitic effects. At present, research on antimicrobial peptides mainly focuses on their potential as alternatives to antibiotics, especially in the face of the increasingly serious antibiotic resistance. Unlike traditional antibiotics, antimicrobial peptides have multiple mechanisms of action, such as direct destruction of bacterial cell membranes, inhibition of biofilm formation and immune regulation. They have good inhibitory effects on a variety of drug-resistant bacteria and are not easy to induce bacterial resistance, which has significant advantages. Due to their low toxicity and good biocompatibility, antimicrobial peptides show broad prospects in clinical applications.

[0004] At present, antimicrobial peptides have been applied in many fields, including medicine, agriculture, food preservation and aquaculture. In the field of medicine, antimicrobial peptides are being developed for the treatment of systemic infections, skin infections, wound healing and oral care. However, antimicrobial peptides still face challenges in practical applications, such as poor in vivo stability, high cost, production difficulties, potential toxicity and immunogenicity. In order to overcome these obstacles, researchers are exploring strategies such as structural optimization, synthetic modification and delivery system improvement to enhance the stability and safety of antimicrobial peptides and reduce production costs. In the future, with the advancement of technology and in-depth research, antimicrobial peptides are expected to play a more important role in the treatment of antimicrobial infections.

[0005] SAAP-148 is an artificially designed antimicrobial peptide derived from the natural antimicrobial peptide LL-37. The antimicrobial activity and stability are enhanced by improving the amino acid sequence. SAAP-148 can effectively fight against a variety of drug-resistant pathogens, including MRSA, CRE and VRE. Its mechanism of action is mainly through destroying the integrity of the bacterial cell membrane, resulting in leakage of cell contents and bacterial death. Studies have shown that SAAP-148 has strong penetration into bacterial biofilms and can be used to treat difficult-to-heal chronic wound infections. In addition, SAAP-148 shows low toxicity and good biocompatibility in vitro and in animal models. Due to its potent antibacterial effect and relative safety, SAAP-148 is considered to be a promising new antibacterial therapeutic candidate that is expected to be used to combat antibiotic-resistant pathogen infections.

[0006] In view of the current treatment status of severe pneumonia, sepsis and skin wound infections caused by drug-resistant bacteria, there is still an urgent need to develop more efficient and less toxic therapeutic antimicrobial peptides. Summary of the invention

[0007] In view of the deficiencies in the prior art, the present invention provides an antimicrobial peptide having the effect of killing drug-resistant bacteria and its application. The antimicrobial peptide AMP-36 has potential activity of killing broad-spectrum drug-resistant bacteria.

[0008] The technical solution of the present invention is as follows:

[0009] An antimicrobial peptide AMP-36, whose amino acid sequence is: LKRVWKRVFKLLKRYWRQLKKPVRRAGLQFPVGRLR, as shown in SEQ ID NO.1.

[0010] Preferably according to the present invention, the antimicrobial peptide AMP-36 has the effect of killing drug-resistant bacteria.

[0011] Further preferably, the drug-resistant bacteria include: methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Klebsiella pneumoniae (CRKP), carbapenem-resistant Escherichia coli (CRE), and pan-drug-resistant Acinetobacter baumannii (MDR-AB).

[0012] Further preferably, the minimum inhibitory concentration of the antimicrobial peptide AMP-36 against methicillin-resistant Staphylococcus aureus (MRSA) is 16 μg / mL.

[0013] Further preferably, the minimum inhibitory concentration of the antimicrobial peptide AMP-36 against carbapenem-resistant Klebsiella pneumoniae (CRKP) is 16 μg / mL.

[0014] Further preferably, the minimum inhibitory concentration of the antimicrobial peptide AMP-36 against carbapenem-resistant Escherichia coli (CRE) is 4 μg / mL.

[0015] Further preferably, the minimum inhibitory concentration of the antimicrobial peptide AMP-36 against pan-drug resistant Acinetobacter baumannii (MDR-AB) is 4 μg / mL.

[0016] Further preferably, the antimicrobial peptide AMP-36 can cause damage to the cell membrane of drug-resistant bacteria.

[0017] The use of the antimicrobial peptide AMP-36 in the preparation of a product having the function of killing drug-resistant bacteria.

[0018] Preferably according to the present invention, the drug-resistant bacteria include: methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Klebsiella pneumoniae (CRKP), carbapenem-resistant Escherichia coli (CRE), and pan-drug-resistant Acinetobacter baumannii (MDR-AB).

[0019] An antimicrobial agent comprising the antimicrobial peptide AMP-36.

[0020] A drug for treating severe pneumonia or sepsis or skin wound infection caused by drug-resistant bacteria or non-drug-resistant bacteria, comprising the above-mentioned antimicrobial peptide AMP-36.

[0021] Preferably according to the present invention, the drug-resistant bacteria include: methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Klebsiella pneumoniae (CRKP), carbapenem-resistant Escherichia coli (CRE), and pan-drug-resistant Acinetobacter baumannii (MDR-AB).

[0022] Preferably according to the present invention, the drug is administered by intravenous injection, aerosol inhalation or local application.

[0023] Beneficial effects:

[0024] 1. The present invention is based on the antimicrobial peptide SAAP-148, and designs a new antimicrobial peptide AMP-36 by adding a partial sequence of the antimicrobial peptide Buforin2b with bacterial nucleus damage effect and deleting a repetitive sequence with membrane-breaking properties. It is unexpectedly found that the antimicrobial peptide AMP-36 has the effect of killing drug-resistant bacteria, and has a stronger ability to kill drug-resistant bacteria and lower toxicity to normal cells than SAAP-148, which lays a foundation for the development of efficient and low-toxic treatment methods for diseases infected by drug-resistant bacteria and non-drug-resistant bacteria and the development of new anti-infective biological drugs. The development of antimicrobial peptide drugs or related products including wound repair dressings has important scientific and economic value in solving major clinical problems - infection, reducing the mortality rate of severe pneumonia, improving the cure rate of bacterial pneumonia, sepsis and infected wounds, and reducing the use rate of antibiotics.

[0025] 2. The novel antimicrobial peptide AMP-36 of the present invention has the characteristics of strong bactericidal effect at low concentrations and a broad-spectrum killing effect. It can provide new biotechnology and methods for the efficient and low-toxic treatment of severe pneumonia, sepsis and skin wound infections caused by resistant and non-resistant bacteria. This study opens up new possibilities for the development of new anti-infective drugs, lays the foundation for the development of corresponding therapeutic products, and provides a feasible approach for clinical medical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a simulated image of the three-dimensional structure of the antimicrobial peptides SAAP-148 and AMP-36.

[0027] Figure 2 This is the time-killing curve of the antimicrobial peptides SAAP-148 and AMP-36 against drug-resistant bacteria.

[0028] Figure 3 This is a scanning electron microscopy image of MRSA after treatment with the antimicrobial peptide AMP-36.

[0029] Figure 4 The results show the hemolytic activity of antimicrobial peptides SAAP-148 and AMP-36 on human erythrocytes. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with embodiments and drawings, but the present invention is not limited by the following embodiments.

[0031] Sources of bacteria:

[0032] Methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Klebsiella pneumoniae (CRKP), carbapenem-resistant Escherichia coli (CRE), and pan-drug-resistant Acinetobacter baumannii (MDR-AB) were all provided by the Laboratory Department of the Second Hospital of Shandong University. Other technicians in this field can purchase the above-mentioned strains from commercial channels to repeat the relevant experiments of the present invention.

[0033] Cryopreservation of bacteria:

[0034] Using the glycerol cryopreservation method, 500 μL of logarithmic phase bacterial solution was mixed with 500 μL of 50% sterile glycerol solution, and the mixture was evenly pipetted to make a bacterial suspension, which was then frozen at -80°C for storage.

[0035] Bacterial recovery and culture:

[0036] Take the glycerol-frozen strain, thaw it quickly at room temperature, then streak or spread it on the nutrient agar plate, culture it in a 37°C constant temperature incubator for 18-24 hours, pick 3-4 colonies with a bacteria loop and add them to the liquid culture medium, and culture it overnight in a shaker at 220 rpm and 37°C until the logarithmic phase.

[0037] Example 1: Design and synthesis of a novel antimicrobial peptide AMP-36

[0038] 1. Design of a novel antimicrobial peptide AMP-36

[0039] The synthetic antimicrobial peptide SAAP-148 from the human cathelicidin family LL-37 was selected as the design basis for the new antimicrobial peptide. Based on the cell membrane-destroying effect of SAAP-148, the antimicrobial peptide Buforin2b with DNA / RNA-binding killing effect was connected, and the repetitive amino acid sequence with membrane-destroying properties was cut off to synthesize the new antimicrobial peptide AMP-36.

[0040] Among them, the amino acid sequence of the antimicrobial peptide AMP-36 is: LKRVWKRVFKLLKRYWRQLKKPVRRAGLQFPVGRLR, SEQ ID NO.1;

[0041] The amino acid sequence of the antimicrobial peptide SAAP-148 is: LKRVWKRVFKLLKRYWRQLKKPVR, SEQ ID NO.2.

[0042] 2. Synthesis and storage of antimicrobial peptides

[0043] The antimicrobial peptides were synthesized by Gill Biochemical Company using the peptide solid phase synthesis method. The lyophilized peptides were stored at -20°C and dissolved as needed.

[0044] The peptide prediction analysis software was used to compare the amino acid sequences of the antimicrobial peptide SAAP-148 and the novel antimicrobial peptide AMP-36. Figure 1 As shown in Table 1, it was found that the new antimicrobial peptide AMP-36 has a richer α-helical structure, a higher positive charge number, and stronger hydrophobicity.

[0045] Table 1. Analysis of properties of antimicrobial peptides SAAP-148 and AMP-36

[0046]

[0047] Example 2: Antibacterial effect of the novel antimicrobial peptide AMP-36

[0048] The minimum inhibitory concentration was used to evaluate the antibacterial effect of antimicrobial peptides. The strains to be tested (MRSA, CRE, MDR-AB, CRKP) were cultured to the logarithmic phase, the bacterial solution was taken, the bacteria were washed twice with PBS buffer, and the concentration of the bacteria was adjusted to 1.5×10 8 CFU / mL, diluted to 1×10 6 CFU / mL. Weigh the lyophilized powder of the antimicrobial peptide to be tested, add sterile double distilled water to fully dissolve it, prepare a stock solution for use, dilute the stock solution with MHB liquid medium to the highest drug concentration of 256μg / mL, and use MHB liquid medium to dilute it in two-fold gradients. Take a sterile 96-well plate, add 100μL of antimicrobial peptide solution with a drug concentration of 128μg / mL to the first column, and add the gradient diluted antimicrobial peptide solution to the subsequent columns in sequence. Add 100μL of the diluted bacterial suspension to each concentration of drug well in turn, and the final bacterial concentration is about 5×10 5 CFU / mL. Set up negative control wells (containing only bacterial suspension without antimicrobial peptides) and positive control wells (containing only culture medium without bacteria). Place the 96-well plate in a 37°C bacterial incubator for 16-18 hours. Measure the bacterial growth in each well by observation and microplate reader (OD450nm), and read the lowest drug concentration without bacterial growth, which is the minimum inhibitory concentration (MIC) of the bacteria to the drug.

[0049] The results are shown in Table 2. Compared with the antibacterial ability of the antimicrobial peptide SAAP-148, the new antimicrobial peptide AMP-36 also has a broad-spectrum antibacterial activity and has the characteristics of strong bactericidal ability at lower concentrations.

[0050] Table 2. Minimum inhibitory concentrations of antimicrobial peptides SAAP-148 and AMP-36 against different drug-resistant bacteria (μg / mL, n=3)

[0051]

[0052] Example 3: Bactericidal speed and duration of action of the novel antimicrobial peptide AMP-36

[0053] The time-kill curve was drawn to detect the bactericidal activity of the antimicrobial peptide. 6 CFU / mL of MRSA bacterial suspension, co-culture the antimicrobial peptide solution and bacterial suspension according to 0, 1 / 2MIC, 1×MIC, 2×MIC, 4×MIC, respectively, and sample at incubation time of 0.5h, 1h, 2h, 4h, 8h, 16h, 24h, respectively, and dilute 10 times after sampling. Take 100μL of the dilution and spread it on the nutrient agar plate, place it in a 37℃ bacterial incubator for 18-24h, and then count the colonies. The colony count is expressed in CFU / mL. The data is Lg CFU / mL as the ordinate and time (h) as the abscissa to make a time-killing curve.

[0054] Time kill curve Figure 2 As shown, the new antimicrobial peptide AMP-36 at MIC concentration can effectively kill the drug-resistant bacteria MRSA, and at an antimicrobial peptide concentration of 16 μg / mL, there is a significant difference in the killing ability of the two antimicrobial peptides, and the new antimicrobial peptide AMP-36 is significantly stronger than the antimicrobial peptide SAAP-148.

[0055] Example 4: Destructive effect of the novel antimicrobial peptide AMP-36 on bacterial membranes

[0056] The damage of antimicrobial peptides to bacteria was observed using a scanning electron microscope. The drug-resistant bacteria MRSA was cultured to the logarithmic phase, and the bacterial solution was taken. The bacteria were washed twice with PBS buffer and resuspended in PBS buffer to prepare a bacterial concentration of 1.5×10 8 CFU / mL bacterial suspension. The new antimicrobial peptide AMP-36 solution was used to treat the above bacterial suspension at 0 (CON group), 1×MIC, and 2×MIC, respectively, incubated at 37°C for 2h, centrifuged and discarded the supernatant, washed twice with PBS buffer, fixed the treated bacteria with glutaraldehyde, rinsed and dehydrated with a series of ethanol gradients, dried, metal-plated, and prepared. The morphological changes of the bacteria were observed with a scanning electron microscope, and the damage of the antimicrobial peptide to the bacterial membrane was analyzed.

[0057] The results are as follows Figure 3 As shown, the bacteria in the CON group had regular morphology and a smooth surface; the group treated with the new antimicrobial peptide AMP-36 at MIC concentration was able to destroy the bacterial membrane of drug-resistant bacteria MRSA, causing depression and damage to the bacterial membrane and deformation of the bacterial body; the effect was more significant at 2×MIC concentration.

[0058] Example 5: Drug toxicity of the novel antimicrobial peptide AMP-36

[0059] The drug toxicity of antimicrobial peptides was tested by human erythrocyte hemolysis. Non-coagulated blood was collected from healthy subjects, centrifuged, and erythrocytes were collected and washed twice. 1 mL of antimicrobial peptide solution with each concentration gradient (0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256 μg / mL) was mixed with 20 μL of the above erythrocyte suspension and incubated in a 37°C incubator for one hour. Centrifuge, take photos, and aspirate 100 μL of supernatant into a 96-well plate. The absorbance of the supernatant was measured with an enzyme reader (OD 540 nm). The complete hemolysis group (treated with ddH2O) was set as the positive control group and the untreated group (treated with PBS buffer) was set as the negative control group. The hemolysis rate was calculated to evaluate the hemolytic activity of the antimicrobial peptides.

[0060] The results are as follows Figure 4 As shown, the hemolytic activity of the new antimicrobial peptide AMP-36 at the effective bactericidal concentration is significantly lower than that of the antimicrobial peptide SAAP-148, and it has better safety.

Claims

1. An antimicrobial peptide AMP-36, characterized in that: Its amino acid sequence is: LKRVWKRVFKLLKRYWRQLKKPVRRAGLQFPVGRLR, as shown in SEQ ID NO.

1.

2. Use of the antimicrobial peptide AMP-36 according to claim 1 in the preparation of a product having the effect of killing drug-resistant bacteria, wherein the drug-resistant bacteria are: methicillin-resistant Staphylococcus aureus, carbapenem-resistant Klebsiella pneumoniae, carbapenem-resistant Escherichia coli, and pan-drug-resistant Acinetobacter baumannii.

3. An antibacterial agent, characterized in that It comprises the antimicrobial peptide AMP-36 as claimed in claim 1.

4. A drug for treating severe pneumonia or sepsis or skin wound infection caused by drug-resistant bacteria or non-drug-resistant bacteria, characterized in that: It comprises the antimicrobial peptide AMP-36 as claimed in claim 1.

5. The drug according to claim 4, characterized in that The drug is administered by intravenous injection, aerosol inhalation or local external application.

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